This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mechanical signals have been shown to regulate various physiological behaviors, including cell growth, differentiation, apoptosis, motility and gene expression. The details of the processes by which mechanical cues result in biochemical and cellular change, or mechanotransduction, are largely unknown. One proposed mechanism is that a force-driven conformational change results in altered binding affinity, essentially converting a mechanical signal into a concentration change. We will characterize properties of force-induced binding changes, through detailed structural and energetic analysis of protein pulling simulations. Methodologically the work uses a multi-dimensional replica exchange protocol, which speeds up convergence through better sampling, and the Weighted Histogram Analysis Method (WHAM) to compute free energy changes induced by mechanical perturbation. We are focusing our efforts on the focal adhesion targeting (FAT) domain of focal adhesion kinase binding to a paxillin peptide. Focal adhesions, the cell anchor points to the extracellular environment, are dynamical protein complexes known to act as mechanosensory devices, where mechanical forces can regulate the assembly of the site and trigger signaling. While the precise identity of proteins responsive to force is not elucidated, interactions between FAT and paxillin play an important role in focal adhesion formation and are thought to be part of the mechanosensing machinery. Preliminary simulations have shown that force can induce strengthening of FAT-paxillin binding interactions through activation of new contacts. We plan to investigate variable effects along different pulling directions to gain insight into the robustness of this response in a biological context, as well as to carry out mutational studies of force-bearing residues. Detailed understanding of the molecular mechanisms of mechanotransduction could lead to advances in therapeutics and tissue engineering, with design of culture conditions mimicking the cells natural chemical and mechanical environment.